Project Summary/Abstract RNA plays key roles in cells and organisms including as a carrier of protein-coding information and as a regulator of gene expression. RNA therapeutics and RNA-based vaccines, which exploit the natural functions of RNA in creating physiological responses designed to prevent or treat disease, have received increasing attention in the past few years, particularly for developing novel vaccines and for curing rare diseases caused by heritable genetic defects. The expanded effort in RNA therapeutics and RNA vaccines has created a new demand for RNA molecules manufactured in large quantities to precise specifications. In particular, the need to create RNA molecules >1kb in length and to incorporate modified nucleotides for more efficient delivery, higher stability and better clinical efficacy, has compounded this manufacturing problem. Although RNAs have been produced enzymatically in vitro for several decades with the use of bacteriophage RNA polymerases, the enzymes traditionally used to produce RNA for R&D purposes are not suited for the demanding specifications that apply to RNA molecules intended for RNA therapeutics. A new class of enzymes, highly optimized for synthesis of long RNAs with specific sequences and structures, need to be created to meet this new demand. In this project, Primordial Genetics aims to express, purify and characterize known but so-far untested single-subunit RNA polymerases that can be used as starting reagents and genetic building blocks in the development of specialized RNA manufacturing enzymes. We will test 50 different enzymes, representing the natural diversity of bacteriophage RNA polymerases, for their ability to meet the critical requirements for in vitro RNA synthesis, including efficient, high- yield RNA synthesis, incorporation of non-natural nucleotides and high RNA quality. The two best enzymes will be improved by mutagenesis based on structural modeling, using the structural and functional information available for this class of enzymes. The proposed work is a feasibility study for isolating and developing novel enzymes suitable for RNA manufacturing, and also for creating an enzyme development pipeline that can meet the varied needs for manufacturing a diversity of RNA sequences, sizes and chemical structures represented in RNA vaccines and RNA therapeutic products under development. The enzymes discovered and improved in this work will be directly useful for RNA manufacturing applications, and can be licensed or sold to companies developing RNA vaccines and therapeutics as well as companies building RNA manufacturing capabilities.